The proposed research is designed to determine the role that rhythmic spontaneous electrical activity plays n the development of spinal motor circuits required for normal locomotion. In-ovo drug induced alterations in the frequency of such activity in chick embryos from stages 20-23 was recently shown to alter the expression of specific adhesion/guidance molecules such as PSA-NCAM, EphA4, and CRYPD,to cause alterations in axon fasciculation, and to produce motor axon pathfinding errors. The mechanisms underlying these alterations, including changes in the bursting frequency versus signaling via specific transmitter systems (ACh, GABA etc.), will be elucidated by combining in-ovo application of specific receptor antagonists with exogenous electrical stimulation at different frequencies. Cultured vibratome slices will be used to further define the intracellular signaling cascades that link rhythmic activity to the expression of specific molecules required for proper circuit formation. Ca2+ imaging in these slices will determine how soon newly generated motoneurons and interneurons become incorporated into functional circuits and thus subject to activity dependent regulation. The specific parameters of the activity used and the downstream signaling pathwaysthat are activated will be determined. Attempts to restore motor function after spinal cord injury by either the regeneration of descending inputs or by the exogenous activation of existing circuits will benefit by the more complete understanding of the effects of activity on the formation and maintenance of cord circuits that these studies will provide. The proposed research should be relevant to causing the differentiation of specific motor and interneuron subtypes from stem cells. It will also identify defects in human fetal spinal cord development that could result from maternal ingestion of nicotine as well as other medically prescribed drugs that alter cholinergic or GABAergic transmission.